Biology subjects

Liu, X.

Publications and source records attributed to Liu, X..

At least 595 records · Page 33Linked to original sources

bioRxiv2024

Characterizing Tyrosine Ring Flips in Proteins by 19F NMR

Aromatic ring-flip dynamics are hallmarks of concerted protein "breathing" motions that are essential for biological function. Ring flips occur on a broad range of timescales (ns-s) and have been primarily studied by NMR spectroscopy, typically requiring expensive isotope labeling of aromatic side chains, thereby limiting such studies to a few proteins. Here, we report two novel di-fluorotyrosine probes, 3,5-F2Y and 2,6-F2Y, that can be incorporated into proteins cost-effectively for characterizing and modulating tyrosine ring-flip dynamics. We show that 19F rotating-frame (R1{rho}) relaxation dispersion is powerful for quantifying ring flips on the {micro}s-ms timescale. Importantly, the tyrosine ring-flip rates (kflip) for 3,5-F2Y in GB1 and HPr are comparable to those measured by 1H and 13C NMR studies, validating 3,5-F2Y as a largely non-perturbing, native-like ring-flip probe. In contrast, 2,6-F2Y acts as an effective "brake" on ring flips, enabling direct visualization and quantitative characterization of previously undetected tyrosine ring flips in ubiquitin via 19F lineshape analysis. Furthermore, we use 2,6-F2Y to assess the environmental effects on ring-flip dynamics in crowding reagents and in living X. laevis oocytes. Collectively, our study opens a new avenue for measuring and modulating ring-flip dynamics in vitro and in living cells. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/630691v2_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@dcf8e1org.highwire.dtl.DTLVardef@1578887org.highwire.dtl.DTLVardef@1c481d0org.highwire.dtl.DTLVardef@268ae0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Dorsolateral prefrontal cortex TMS evokes responses in the subgenual anterior cingulate cortex: Evidence from human intracranial EEG

Transcranial magnetic stimulation combined with intracranial local field potential recordings in humans (TMS-iEEG) represents a new method for investigating electrophysiologic effects of TMS with spatiotemporal precision. We applied TMS-iEEG to the dorsolateral prefrontal cortex (dlPFC) in two subjects and demonstrate evoked activity in the subgenual anterior cingulate cortex (sgACC). This study provides direct electrophysiologic evidence that dlPFC TMS, as targeted for depression treatment, can modulate brain activity in the sgACC.

neuroscience↗

bioRxiv2024

Integrative analysis of angiogenic signaling in obesity: capillary features and VEGF binding kinetics

Obesity is a global health crisis, with its prevalence particularly severe in the United States, where over 42% of adults are classified as obese. Obesity is driven by complex molecular and tissue-level mechanisms that remain poorly understood. Among these, angiogenesis--primarily mediated by vascular endothelial growth factor (VEGF-A)--is critical for adipose tissue expansion but presents unique challenges for therapeutic targeting due to its intricate regulation. Systems biology approaches have advanced our understanding of VEGF-A signaling in vascular diseases, but their application to obesity is limited by scattered and sometimes contradictory data. To address this gap, we performed a comprehensive analysis of the existing literature to synthesize key findings, standardize data, and provide a holistic perspective on the adipose vascular microenvironment. The data mining revealed five key findings: (1) obesity increases adipocyte size by 78%; (2) vessel density in adipose tissue decreases by 51% in obese mice, with vessels being 47-58% smaller and 4-9 times denser in comparison with tumor vessels; (3) capillary basement membrane thickness remains similar regardless of obesity; (4) VEGF-A shows the strongest binding affinity for VEGFR1, with four times stronger affinity for VEGFR2 than for NRP1; and (5) binding affinities measured by radioligand binding assay and surface plasmon resonance (SPR) are significantly different. These consolidated findings provide essential parameters for systems biology modeling, new insights into obesity-induced changes in adipose tissue, and a foundation for developing angiogenesis-targeting therapies for obesity.

bioengineering↗

bioRxiv2024

A Systematic Comparison of Single-Cell Perturbation Response Prediction Models

Predicting single-cell transcriptional responses to perturbations is central to dissecting gene regulation and accelerating therapeutic design, yet the field lacks a rigorous, task-spanning assessment of model behavior. We present a large-scale benchmark of 13 representative methods and baselines across 25 datasets spanning diverse perturbation modalities and species, including two primary immune-cell drug-response resources. We evaluated three core tasks--generalization to unseen single-gene perturbations, prediction of combinatorial interactions, and transfer across cell types--using 24 metrics covering expression-level accuracy, relative changes, differential expression recovery, and distributional similarity. Across tasks, performance depended strongly on perturbation effect size and evaluation perspective: expression-level agreement was highest for small-effect perturbations resembling controls, whereas delta- and DE-based metrics improved with larger effects, providing clearer signals. Models shared a conservative bias, with fine-tuned foundation models compressing variance and underestimating synergistic effects in combinations. PerturbNet showed superior recovery of DE signatures in Tasks 1 and 2, while no method consistently generalized across cell types in Task 3, where biological consistency dominated outcomes. This benchmark establishes current methodological limits, clarifies that different metrics probe distinct biological signals rather than redundant summaries of the same prediction problem, and provides a foundation for developing virtual-cell models that more faithfully capture heterogeneous perturbation responses. Teaser: A large-scale benchmark reveals strengths and limits of models predicting single-cell perturbation responses.

bioinformatics↗

bioRxiv2024

Non-invasive diagnosis of early chronic colitis cancerization via amplified sensing of miRNA-21 in NIR-IIb window

Early diagnosis of the colitis-associated colorectal cancer (CRC) is of great significance for improving prognosis and survival rates. However, the clinical used colonoscopy and biopsy methods are invasive and lack of sensitivity at early stage of cancerization. Herein, we present an amplified sensing strategy in the second near-infrared b (NIR-IIb, 1500-1700 nm) window for non-invasive in situ visualization of early cancerization biomarker miRNA-21. A CRC nanosensor composed of Er3+ doped lanthanide nanoparticles, DNAzyme, and IR820 dye is designed as a NIR-IIb ratiometric luminescence reporter, providing rapid feedback and high sensitive miRNA-21 detection with limit of detection (LOD) of 1.26 pM. This strategy enables a non-invasive detection of colitis-associated cancerization up to [~]4 weeks ahead of the clinically used biopsy analysis, providing a promising alternative for early diagnosis of chronic colitis cancerization and guidance for therapeutic management.

bioengineering↗

bioRxiv2024

Multiomic foundation model predicts epigenetic regulation by zero-shot

Deciphering cell type-specific perturbation effects on genes and cellular states demands considerable experimental resources. To overcome this challenge, we propose Perturbation Transformer (PertFormer), a foundation model to uncover functional and regulatory mechanisms through interpretable in silico perturbation process. PertFormer comprises 3 billion parameters pretrained on both bulk and single-cell datasets covering 9 types of multiomics (55 billion bp bulk multiomics and the largest-to-date 1.5 billion paired multiomic samples from 1 million single cells), capturing regulation of 300 kb around every genic region. PertFormer enables zero-shot prediction of functional perturbations and cell fate dynamics, outperforming existing methods by 20.9%-480.2%. PertFormer identifies novel tumor treatment targets, validated experimentally. The generalization capabilities of PertFormer have the potential to accelerate the discovery of biological and clinical targets.

genomics↗

bioRxiv2024

KIF18A in HCC Metastasis: Dual Role in Anoikis Resistance and Chromosome Instability

The study explores the role of KIF18A, a gene linked to whole-genome doubling (WGD) and chromosomal instability (CIN), in the metastatic progression of hepatocellular carcinoma (HCC). We found that KIF18A is a critical link between CIN and anoikis resistance, key factors in HCC metastasis. Through the bulk-seq analysis, we identified the pressures of anoikis and CIN faced by tumor cells during HCC metastasis and identified hub genes central to this process. Our results reveal that E2F activation during HCC progression leads to the transcription of KIF18A, promoting the survival and metastasis of CIN tumors. Overexpression of KIF18A leads to longer tumor life, more micronuclei, and increased survival and metastasis through non-canonical NF-kB activation. Deletion of KIF18A stabilizes the nuclear membrane of the micronucleus, silences cGAS-STING and PI3K-AKT pathways, and inhibits classical NF-kB. The studys limitations include the need for further animal studies to validate the findings and explore the transient activation of the cGAS-STING pathway. Additionally, future research could focus on the potential therapeutic implications of targeting KIF18A in cancer treatment.

cancer biology↗

bioRxiv2024

I2SIM: Boosting High-Fidelity Isotropic Super-Resolution with Image Interference and Spatial-Spectrum Optimization

Spatial resolution is crucial for imaging subcellular structures. The advent of three-dimensional structured illumination microscopy (3D-SIM) greatly benefits the biology community, providing a powerful tool for imaging organelles with a two-fold resolution enhancement in all three dimensions. However, the axial resolution of 3D-SIM is limited to around 300 nm, which is inferior to its lateral resolution. Here, a novel method called image interference SIM (I2SIM) is reported, which utilizes two oppositely positioned objectives to detect fluorescence emission interference under three-beam excitation. By incorporating spectral modulation and spatial domain Frobenius-Hessian optimization, I2SIM achieves an axial resolution approximately twice that of 3D-SIM, reaching around 130 nm. Furthermore, the potential of I2SIM for imaging subcellular structures is demonstrated on various biological samples, including microtubules, actin filaments, and mitochondrial outer membranes. The enhanced optical sectioning capability can be utilized to resolve axial structures that are challenging to discern using ordinary 3D-SIM.

biophysics↗

bioRxiv2024

Spatial Transcriptomics Reveals Expression Gradients in Developing Wheat Inflorescences at Cellular Resolution

The diversity of plant inflorescence architectures is specified by gene expression patterns. In wheat (Triticum aestivum), the lanceolate-shaped inflorescence (spike) is defined by rudimentary spikelets at the base which initiate first but subsequently lag in development compared with central spikelets. While previous studies identified gene expression differences between central and basal inflorescence sections, the spatio-temporal dynamics and gradients along the apical-basal axis remain poorly resolved due to bulk tissue-level techniques. Here, using spatial transcriptomics, we profiled 200 genes across four stages of wheat inflorescence development to cellular resolution. Cell segmentation and unsupervised clustering identified 18 expression domains and their enriched genes, revealing dynamic spatio-temporal organisation along the apical-basal axis of the inflorescence. Along this axis, we uncovered distinct and spatially coordinated gene expression gradients patterning meristems prior to the visible delay in basal spikelet development. This study demonstrates the potential for spatial transcriptomics time-series to advance plant developmental biology.

plant biology↗

bioRxiv2024

Isobaric Tagging and Data Independent Acquisition as Complementary Strategies for Proteome Profiling on an Orbitrap Astral Mass Spectrometer

Comprehensive global proteome profiling that is amenable to high throughput processing will broaden our understanding of complex biological systems. Here, we evaluated two leading mass spectrometry techniques, Data Independent Acquisition (DIA) and Tandem Mass Tagging (TMT), for extensive protein abundance profiling. DIA provides label-free quantification with a broad dynamic range, while TMT enables multiplexed analysis using isobaric tags for efficient cross-sample comparisons. We analyzed 18 samples, including four cell lines (IHCF, HCT116, HeLa, MCF7) under standard growth conditions, in addition to IHCF treated with two H2O2 concentrations, all in triplicate. Experiments were conducted on an Orbitrap Astral mass spectrometer, employing Field Asymmetric Ion Mobility Spectrometry (FAIMS). Despite utilizing different acquisition strategies, both the DIA and TMT approaches achieved comparable proteome depth and quantitative consistency, with each method quantifying over 10,000 proteins across all samples, with slightly more protein-level precision for the TMT strategy. Relative abundance correlation analysis showed strong agreement at both peptide and protein levels. Our findings highlight the complementary strengths of DIA and TMT for high-coverage proteomic studies, providing flexibility in method selection based on specific experimental needs.

biochemistry↗

bioRxiv2024

Artificial Embodied Circuits Uncover Neural Architectures of Vertebrate Visuomotor Behaviors

All brains evolve within specific sensory and physical environments1. Traditionally, neuroscience has focused on studying neural circuits in isolation, yet holistic characterization of their function requires integrative brain-body testing2,3. To investigate the neural and biomechanical mechanisms of sensorimotor transformations, we constructed realistic neuromechanical simulations (simZFish) of the larval zebrafish optomotor response, a visual stabilization behavior4,5. By computationally reproducing the body, physical body-water interactions, visual environments, and experimentally derived neural architectures, we closely replicated the behavior of real zebrafish6. Through systematic manipulation of physiological and circuit features, impossible in biological experiments, we demonstrate how embodiment shapes neural circuit architecture and behavior. When challenged with novel visual stimuli, simZFish predicted neuronal response types, which we identified via calcium imaging in the brain of real zebrafish and used to update the simZFish neural network. In virtual rivers, simZFish performed rheotaxis by using current-induced optic flow as navigational cues, compensating for the simulated water flow. Finally, a physical robot (ZBot) validated the role of embodied sensorimotor circuits in maintaining position in a real river with complex fluid dynamics and visual environments. Together, by iterating between simulations, behavioral observations, neural imaging, and robotic testing, we demonstrate the power of an integrative approach to investigating sensorimotor processing. Research HighlightsO_LIDeveloped simZFish, an open-source neuromechanical simulation modeling the zebrafish optomotor response (OMR). C_LIO_LIDemonstrated that simZFish visuomotor neural circuits are sufficient to maintain position in virtual water currents without additional sensory modalities. C_LIO_LIInvestigated how variations in eye geometry influence neural circuit functionality and behavior. C_LIO_LIConducted optic flow analysis of simulated visual input to identify retinal connectivity requirements, uncovering the factors that shape pretectal neurons preference for the lower posterior visual field. C_LIO_LIsimZFish predicted new neural response types confirmed via real zebrafish calcium imaging. C_LIO_LIValidated simZFish circuits with a physical robot, ZBot, performing rheotaxis in a natural river with rich visual and complex flow dynamics compared to idealized lab and simulation experiments. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/629427v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@11f2acforg.highwire.dtl.DTLVardef@133b1f3org.highwire.dtl.DTLVardef@e808bcorg.highwire.dtl.DTLVardef@1959616_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractCombining data-driven neuromechanical simulations and robotic testing, we established an integrative framework for investigating embodied neural circuit functions. Leveraging the behavioral, neurobiological, and theoretical foundations of the visually guided optomotor response in larval zebrafish (blue, left), we developed a virtual zebrafish, simZFish (red, middle), to replicate realistic hydrodynamic, anatomical, sensory, neural, and behavioral aspects, permitting the investigation of emergent properties driven by embodiment. Validating these findings using a physical, zebrafish-inspired robot, ZBot (grey, right), in naturalistic environments informs further experiments, hypotheses, and robotic design.

neuroscience↗

bioRxiv2024

Ongoing loss of viable neurons for weeks after mild perinatal hypoxia-ischemia

Mild hypoxic-ischemic encephalopathy is common in neonates with no evidence-based therapies, and 30-40% of patients experience adverse outcomes. The nature and progression of mild injury is poorly understood. Thus, we studied the evolution of mild perinatal brain injury using longitudinal two-photon imaging of transgenic fluorescent proteins as a novel readout of neuronal viability and activity at cellular resolution. In vitro, perinatal murine organotypic hippocampal cultures underwent 15-20 minutes of oxygen-glucose deprivation. In vivo, mild hypoxia-ischemia was completed in post-natal day 10 mouse pups of both sexes with carotid ligation and 15 minutes of hypoxia. Consistent with a mild injury, minimal immediate neuronal death was seen and there was no volumetric evidence of injury by ex vivo MRI 2.5 weeks after injury. In both the hippocampus and neocortex, these mild injuries resulted in a significantly delayed and progressive neuronal loss in the second week after injury, measured by fluorophore quenching. Mild hypoxia-ischemia transiently suppressed cortical network activity followed by normal maturation. No post-injury seizures were seen. The participation in network activity of individual neurons destined to die was indistinguishable from those that survived for 4 days post-injury. In conclusion, our results showed that mild perinatal brain injury resulted in a prolonged increase of neuronal death. Neurons that died late were functioning normally for days after injury, suggesting a new pathophysiology of neuronal death. Critically, the neurons destined to die late demonstrated multiple biomarkers of viability long after mild injury, suggesting their later death may be modified with neuroprotective interventions. SIGNIFICANCE STATEMENTNeonatal encephalopathy due to peripartum hypoxia-ischemia (HI) is a major cause of neonatal mortality and morbidity worldwide. Of these infants, most are categorized as having mild HI. Infants with mild HI have significant long-term disabilities. There are currently no evidence-based therapies, largely because the progression and pathophysiology of mild injury is poorly understood. We have identified, for the first time, that mild perinatal HI results in a delayed and prolonged increase in neuronal death. The cortical and hippocampal neurons that die over a week after injury participate normally in neural network activity and exhibit robust viability for many days after injury, indicating a novel pathophysiology of neuronal death. Clinically, these data suggest an extended therapeutic window for mild perinatal HI.

neuroscience↗

bioRxiv2024

Transposable elements drive regulatory and functional innovation of F-box genes

Protein domains of transposable elements (TEs) and viruses increase the protein diversity of host genomes by recombining with other protein domains. By screening 10 million eukaryotic proteins, we identified several domains that define multi-copy gene families and frequently co-occur with TE/viral domains. Among these, a Tc1/Mariner transposase helix-turn-helix (HTH) domain was captured by F-box genes in the Caenorhabditis genus, creating a new class of F-box genes. For specific members of this class, like fbxa-215, we found that the HTH domain is required for diverse processes including germ granule localisation, fertility, and thermotolerance. Furthermore, we provide evidence that HSF-1 mediates the transcriptional integration of fbxa-215 into the heat-shock response by binding to Helitron TEs directly upstream of the fbxa-215 locus. The interactome of HTH-bearing F-box factors suggests roles in post-translational regulation and proteostasis, consistent with established functions of F-box proteins. Based on AlphaFold2 multimer proteome-wide screens, we propose that the HTH domain may diversify the repertoire of protein substrates that F-box factors regulate post-translationally. We further demonstrate that F-box genes repeatedly and independently captured TE domains throughout eukaryotic evolution, and describe an additional instance in zebrafish. In conclusion, we identify recurrent TE domain captures by F-box genes in eukaryotes and provide insights into how these novel proteins are integrated within host gene regulatory networks.

evolutionary biology↗

bioRxiv2024

TMEM55A-mediated PI5P signaling regulates α-cell actin depolymerization and glucagon secretion

Diabetes is associated with the dysfunction of glucagon-producing pancreatic islet -cells, although the underlying mechanisms regulating glucagon secretion and -cell dysfunction remain unclear. While insulin secretion from pancreatic {beta}-cells has long been known to be partly controlled by intracellular phospholipid signaling, very little is known about the role of phospholipids in glucagon secretion. Here we show that TMEM55A, a lipid phosphatase that dephosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-5-phosphate (PI5P), regulates -cell exocytosis and glucagon secretion. TMEM55A knockdown in both human and mouse -cells reduces exocytosis at low glucose, and this is rescued by the direct reintroduction of PI5P. This does not occur through an effect on Ca2+ channel activity, but through a re-modelling of cortical F-actin dependent upon TMEM55A lipid phosphatase activity which occurs in response to oxidative stress. In summary, we reveal a novel pathway by which TMEM55A regulates -cell exocytosis by manipulating intracellular PI5P level and the F-actin network.

cell biology↗

bioRxiv2024

Dual roles of IQCG, a novel microtubule nucleation factor rapidly evolving in humans

The centrosome serves as the main microtubule-organizing structure, playing crucial roles in animal development. Uncovering new components and elucidating their functions helps to understand the regulation of cell maintenance and division by the centrosome. In this study, through a combination of genetic analyses and cell biological characterizations, we identify IQ Motif Containing G (IQCG) as a novel microtubule nucleation factor with a dual role involved in both centrosomal microtubule organization and mitotic spindle formation. IQCG interacts with GSK3{beta} via its N2 region, enhancing its centrosomal accumulation in interphase; subsequently, IQCG disperses into the spindle to promote microtubule generation during mitosis and ensure robust cell division. Furthermore, our analyses indicate that the IQCG gene has undergone accelerated evolution within the human lineage, with positive selection acting on the variable N2 region. This adaptation may confer advantages to human cells by augmenting central regulatory control over cellular activities through the centrosome. Our findings offer novel insights into the functional mechanisms and evolutionary significance of the human centrosome associated with human evolution. Summary blurbThe cellular functions of the novel microtubule nucleation factor IQCG, which enhances centrosomal microtubule organization and cell division robustness, are implicated in human evolution.

cell biology↗

bioRxiv2024

Integrative Omics reveals genetic basis and TaMYB7-A1's function in wheat WUE and drought resilience

Improving water use efficiency (WUE) and drought resistance in wheat is critical for ensuring global food security under changing climate conditions. Here, we integrated multi-omic data, including population-scale phenotyping, transcriptomics, and genomics, to dissect the genetic and molecular mechanisms underlying WUE and drought resilience in wheat. Genome-wide association studies (GWAS) revealed 8,135 SNPs associated with WUE-related traits, identifying 258 conditional and non-conditional QTLs, many of which co-localized with known drought-resistance genes. Pan-transcriptome analysis uncovered tissue-specific expression patterns, core and unique gene functions, and dynamic sub-genomic biases in response to drought. eQTL mapping pinpointed 146,966 regulatory loci, including condition-specific hotspots enriched for genes involved in water regulation, osmoregulation, and photosynthesis. Integration of Weighted gene co-expression network analysis (WGCNA), Summary-data-based Mendelian Randomization (SMR) and GWAS, eQTLs identified 207 candidate causal genes as key regulators for WUE-related traits in wheat, such as TaMYB7-A1. Functional analyses found that TaMYB7-A1 enhances drought tolerance by promoting root growth, reducing oxidative stress, and improving osmotic regulation, enabling better water access and survival under stress. It also increases photosynthesis efficiency and WUE, boosting yield under drought without compromising performance in well-watered conditions, making it ideal target for breeding. Our findings provide a comprehensive omic framework for understanding the genetic architecture of WUE and drought resistance, offering valuable targets for breeding resilient wheat varieties.

plant biology↗

bioRxiv2024

Genome-enabled stilbenoid gene cluster discovery in Pleuropterus multiflorus

Stilbenoids, a class of compounds with the stilbene skeleton as part of plant secondary metabolism, are renowned for their diverse health benefits. Among them, 2,3,5,4'-Tetrahydroxystilbene-2-O-{beta}-D-glucoside (THSG), specifically abundant in the famous medicinal plant Pleuropterus multiflorus, exhibits significant pharmacological properties. A selfing progeny of P. multiflorus commercial variety Jinwufugui No.1, which contains high THSG content, was used for genome sequencing. Through the combination of weighted correlation network analysis, genome mining, and enzymatic characterization studies, we identified an unpredicted biosynthetic gene cluster responsible for THSG biosynthesis. This cluster includes a stilbene synthase (PmSTS1), a flavin-containing monooxygenase (PmFMO3), and a UDP-glycosyltransferase (UGT72B90). Our findings suggest this stilbenoid gene cluster is formed by transposable element-mediated gene duplications and neofunctionalization, and regulated by MYB and NAC type transcription factors. Notably, PmFMO3, as the first identified stilbenoid-2-hydroxylase, was confirmed that it can modify the other stilbenoids without the A ring modifications. The novel type of gene cluster, in which FMO serves as the key decorating enzyme, will guide the identification of more biosynthetic gene clusters in plants. In addition, the characterization of PmFMO3 extends our understanding of the FMO gene function in plants, and provides an important catalytic bioparts for the heterologous synthesis of polyhydroxylated stilbenoids.

molecular biology↗

bioRxiv2024

Zero valent sulfur is the product of assimilatory sulfite reductase and a substrate of cysteine synthase

The long process of the assimilatory sulfate-reduction consists of sulfate reduction to H2S and then H2S assimilation for cysteine synthesis. However, recent studies indicate that both H2S and zero valent sulfur (S0) are produced during dissimilatory sulfate reduction process, which leads to the question whether S0 also is involved in assimilatory sulfate-reduction. Herein, phylogenetic analysis grouped assimilatory sulfite reductases into five clusters and we re-examined four representatives from two major clusters. These enzymes produced both S0 and H2S from sulfite. However, S0 was more like a direct product, while H2S was just a derivative of S0 via chemical reduction. Phylogenetic analysis of cysteine synthases grouped them into six clusters, and we re-examined six representatives from two major clusters. These enzymes used both glutathione persulfide (GSSH, a common intracellular compound containing a S0 atom) and H2S as substrates to synthesize cysteine, but preferred to use GSSH. These findings indicated that S0, other than H2S, is the key intermediate during the process of inorganic sulfur converting to organic sulfur. ImportanceSulfate reduction is an important link in the global sulfur cycle. Assimilatory sulfate-reduction consists of sulfate-reduction to H2S and then H2S assimilation for cysteine synthesis. This concept was founded in 1950s and has not been challenged until today. In this study, we re-examined four representative assimilatory sulfite reductases (aSiRs) and found that these enzymes directly reduced sulfite to zero valent sulfur (S0). A small portion of produced S0 was reduced to H2S via aSiR-independent chemical reactions. Further, we re-examined six representative cysteine synthases and found that these enzymes used S0 derivative compounds (thiosulfate and glutathione persulfide) as substrates to synthesize cysteine. Thus, S0 is the key intermediate of assimilatory sulfate reduction process, other than H2S.

microbiology↗